Volume 129, Issue 3, Pages 617-631 (May 2007) An Evolutionarily Conserved Mechanism for MicroRNA-223 Expression Revealed by MicroRNA Gene Profiling  Taro Fukao, Yoko Fukuda, Kotaro Kiga, Jafar Sharif, Kimihiro Hino, Yutaka Enomoto, Aya Kawamura, Kaito Nakamura, Tsutomu Takeuchi, Masanobu Tanabe  Cell  Volume 129, Issue 3, Pages 617-631 (May 2007) DOI: 10.1016/j.cell.2007.02.048 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Mouse Pri-miR-223 Identified by Genome-Wide Screening of Pri-miRNAs (A) Schematic for identification of cDNAs encoding known pre-miRNA sequences with conserved upstream genomic regions in FANTOM3. (B) Two cDNAs, AK036748 and AK170206, encoding murine miR-223 precursor sequences (upper panel) and their gene structures (lower). The numbers denote the corresponding position at the Mus musculus genomic clone, RP23-23B17. (C) Conservation of pri-miR-223 5′ proximal genomic region (upper). A luciferase reporter assay was performed in RAW264.7 cells with indicated reporters (lower). 1 indicates ppri-mmiR-223−758-Luc, 2 indicates ppri-mmiR-223−476-Luc, 3 indicates ppri-mmiR-223−233-Luc, 4 indicates ppri-mmiR-223−146-Luc, 5 indicates ppri-mmiR-223−77-Luc, and 6 indicates pGL3 basic. (D) Upper panel: The 5′ proximal promoter region of mouse pri-miR-223 with putative binding motifs for several transcription factors. The transcription start site of AK036748 is fixed as +1. Lower panel: Highly conserved genomic site that contains PU.1- and C/EBP-binding motifs. Cell 2007 129, 617-631DOI: (10.1016/j.cell.2007.02.048) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 PU.1 and C/EBPβ Activate Mouse Pri-miR-223 Promoter (A) Interactions of PU.1 and C/EBPβ with the core promoter region of mouse pri-miR-223 were examined in WEHI-3 cells using ChIP assay as described in Experimental Procedures. As controls, Cathepsin C promoter (positive control) and c-Myc promoter (negative control) were assayed. Two percent of input DNA was used as a positive control of PCR. The antibody against MKP-1 was used as negative control for ChIP. (B) A graded amount (1[0.5 μg], 1/3, and 1/9) of each expression vector together with reporter vectors (Firefly Luc (FL): 0.2 μg; Renilla Luc (RL): 0.05 μg) was transfected into NIH3T3 cells and assayed for a luciferase reporter assay. (C) A graded dose (1[0.3μg], 1/3, and 1/9) of C/EBPβ expression vector together with PU.1 expression plasmid (0.2 μg) and reporter vectors (FL: 0.2 μg; RL: 0.05 μg) were transfected in NIH3T3 and examined for the pri-miR-223 promoter activity by a luciferase reporter assay. (D) A graded amount (1[0.5μg], 1/3, and 1/9) of mouse GATA-1 expression vector together with reporter vectors (FL: 0.2 μg; RL: 0.05 μg) was transfected into RAW264.7 cells and examined for pri-miR-223 promoter activity by a luciferase reporter assay. All data are represented as mean ± standard deviation (SD) from triplicate assays. Cell 2007 129, 617-631DOI: (10.1016/j.cell.2007.02.048) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 PU.1 as Well as C/EBP as a Regulator of Pri-miR-223 Transcription (A) The mutant sequences of mouse pri-miR-223 promoter reporter vector are shown (left). The effects of mutations on endogenous pri-miR-223 promoter activity were examined by a luciferase reporter assay. Data are shown as promoter activity relative to the basal endogenous pri-miR-223 promoter activity. (B and C) Repression of the endogenous mouse pri-miR-223 promoter activity by overexpression of dominant-negative PU.1 (DN-PU.1; B) or C/EBPβ (C). Together with reporter vectors (FL: 0.2 μg, RL; 0.05 μg), indicated expression vectors (0.5 μg) were transfected into RAW264.7 cells. The activity of mouse pri-miR-223 promoter was then examined by a luciferase reporter assay. For DN-PU.1 expression, each expression vector was used with two doses (1[0.5μg] or 1/10). Data are represented as mean ± SD from triplicate assays. ∗ indicates p < 0.05 in a Student's t test. (D) The levels of pri-miR-223 and mature miR-223 in RAW264.7 cells transfected with either pcDNA3, DN-PU.1 (PU.1ΔAD), or DN-C/EBPβ were examined by quantitative real-time RT-PCR as detailed in the Experimental Procedures. Transfectants were enriched as described in the Supplemental Data. Data are shown as mean ± SD from triplicate measurements. (E) The level of pri-miR-223 in undifferentiated and differentiated M1 cells was examined by quantitative real-time RT-PCR. Data are shown as mean of triplicate assays. (F) Stable transfectants of M1 cells with either control (pcDNA3) or DN-PU.1 (PU.1ΔAD) expression vector were prepared as described in the Supplemental Data and were cultured in the presence of mrIL-6 for 4 days to induce differentiation. The level of pri-miR-223 in each transfectant was then examined by quantitative RT-PCR. Data are represented as mean ± SD from triplicate tests. Cell 2007 129, 617-631DOI: (10.1016/j.cell.2007.02.048) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Cloning and Characterization of Human Pri-miR-223 in Bone Marrow (A) Detection of the putative human pri-miR-223, LOC389865 mRNA, in bone marrow cDNA by RT-PCR with indicated primers. (B) Cloning of human pri-miR-223. Two alternative splicing forms (Long, L, and Short, S) were cloned as human pri-miR-223, and their genome plots are shown. Numbers denote the position at H. sapiens genomic clone, RP6-159A1 from Xq12-13.3. (C) Cloning of full-length 5′ ends of human pri-miR-223. Full-length transcripts were purified from bone marrow total RNA or HeLa cells and were reverse transcribed with GSP3 to obtain full-length human pri-miR-223 5′ ends. (D) Schematic for positions of the conserved genomic region, identified transcription start sites, and pre-miR-223 sequence (upper panel). Details of sequences around the transcription start sites of human pri-miR-223 are shown (lower panel). Shaded region indicates the conserved site as shown in Figure 1D and contains the core promoter region. Faint letters denote the first intronic sequence of human pri-miR-223. MTSS indicates major transcription start site. Thin arrows indicate the positions of minor transcription start sites. (E) Luciferase reporter assays were performed in either THP-1 or U937 cells using indicated reporters. 1 indicates ppri-hmiR-223−669-Luc, 2 indicates ppri-hmiR-223−293-Luc, 3 indicates ppri-hmiR-223−198-Luc, 4 indicates ppri-hmiR-223−96-Luc, 5 indicates ppri-hmiR-223−16-Luc, and 6 indicates pGL3 basic. Cell 2007 129, 617-631DOI: (10.1016/j.cell.2007.02.048) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 miR-223 Regulation during ATRA-Induced Differentiation of NB4 Cells (A) Transcription start sites of pri-miR-223 in NB4 cells were determined by 5′ RACE. The 5′ RACE products (RPs) from total RNA samples of bone marrow (BM), NB4 cells (None), ATRA-treated NB4 cells, and HeLa cells are shown (upper panel). M1 indicates 100 bp step ladder; M2 indicates 1 kb DNA ladder (G5711, Promega). The transcription start sites indicated by the 5′ RACE products are shown (lower panel). (B) Detection of the human pri-miR-223, LOC389865 mRNA, in ATRA-treated and non-treated NB4 cells using PCR with primer set 3 as indicated in Figure 4A (upper panel). BM indicates bone marrow; ×5 indicates five times more RNA template; and DW indicates distilled water. M1 indicates 100 bp step ladder; M2 indicates 1 kb DNA ladder (G5711, Promega). (Middle panel) The target site of human pri-miR-223 for a specific primer set for real-time PCR is shown. (Lower panel) Upregulation of pri-miR-223 (left) and mature miR-223 (right) during ATRA-induced differentiation of NB4 was observed by quantitative real-time PCR. (C) Potent activation of the conserved pri-hmiR-223 promoter in NB4 cells during ATRA-induced differentiation. Luciferase reporter assay was performed as described in Experimental Procedures. 1 indicates ppri-hmiR-223−669-Luc, 2 indicates ppri-hmiR-223−293-Luc, 3 indicates ppri-hmiR-223−198-Luc, 4 indicates ppri-hmiR-223−96-Luc, 5 indicates ppri-hmiR-223−16-Luc, and 6 indicates pGL3 basic. Data are shown as mean ± SD of triplicate measurements. (D) Effects of synthetic siRNA-mediated PU.1 or C/EBPα knockdown on ATRA-induced pri-miR-223 promoter activity. NC indicates negative control. Data are shown with mean ± SD of triplicate measurements. (E and F) Knockdown of PU.1 (left) or C/EBPα (p42 isoform; right) by corresponding shRNA vectors was confirmed by western blotting (E). Note that there is significant upregulation of PU.1 upon ATRA-treatment as reported previously (Mueller et al., 2006). A portion of each sample was subjected to measurement of the pri-miR-223 level (F). Transfectants were enriched as described in the Supplemental Data. Data in (F) were shown with mean ± SD of triplicate assays. Cell 2007 129, 617-631DOI: (10.1016/j.cell.2007.02.048) Copyright © 2007 Elsevier Inc. Terms and Conditions